Epimerized derivatives of k5 polysaccharide with a very high degree of sulfation

ABSTRACT

A new method is described for the oversulfation of epiK-N sulfate to obtain an epiK5-amine-O-oversulfate with very high sulfation degree which, by subsequent N-sulfation, provides new epiK5-N,O-oversulfate-derivatives with a sulfation degree of at least 4, basically free of activity on the coagulation parameters and useful in the cosmetic or pharmaceutical field. Also described are new low molecular weight epiK5-N-sulfates useful as intermediates in the preparation of the corresponding LMW-epiK5-N,O-oversulfate-derivatives.

OBJECT OF THE INVENTION

The present invention concerns new derivatives of K5 polysaccharide witha very high degree of sulfation, a process for their preparation, highlyO-sulfated new intermediates useful in their synthesis andpharmaceutical compositions containing said derivatives of K5polysaccharide as active ingredients basically free of activity oncoagulation.

In particular, the invention refers to a process for the preparation ofepiK5-N,O-oversulfates starting with a K5 polysaccharide, previouslyN-deacetylated, N-sulfated and C5-epimerized at least 20%, throughO-oversulfation in suitable conditions and subsequent N-sulfation, tosaid epiK5-N,O-oversulfates of antiangiogenetic and antiviral activityand to new low molecular weight intermediates of epi-K5-N-sulfates.

BACKGROUND OF THE INVENTION

The glycosaminoglycans such as heparin, heparan sulfate, dermatansulfate, chondroitin sulfate and hyaluronic acid are biopolymers thatare industrially extracted from various animal organs.

In particular, heparin, mainly obtained by extraction from theintestinal mucous membrane of pigs or bovine lung, is a polydispersedcopolymer with a molecular weight distribution from approximately 3,000to approximately 30,000 D consisting of a chain mixture basicallyconsisting of a uronic acid (glucuronic acid or iduronic acid) and of anamino sugar (glucosamine) linked by α-1→4 or β-1→4 bonds. In heparin,the uronic unit can be O-sulfated in position 2 and the glucosamine unitis N-acetylated or N-sulfated, 6-O-sulfated, and 3-O-sulfated inapproximately 0.5% of the glucosamine units present.

The properties and natural biosynthesis of heparin in mammals have beendescribed by Lindahl et al., 1986 in Lane, D. and Lindahl, U. (Editors)“Heparin. Chemical and Biological Properties; Clinical Applications”,Edward Arnold, London, Pages 159-190, by Lindahl, U, Feingold D. S. andRoden L, 1986 TIBS, 11, 221-225 and by Conrad H. E. “Heparin BindingProteins”, Chapter 2: Structure of Heparinoids. Academic Press, 1998.The biosynthesis of heparin occurs starting with its precursorN-acetyl-heparosan consisting of a chain mixture consisting of therepetitive disaccharide unit glucuronyl-β1-4-N-acetylglucosamine. Saidprecursor undergoes enzymatic modifications which partially hydrolysethe N-acetyl group, substituting it with an SO₃ ⁻ group, epimerize thecarboxyl in position 5 of a part of the glucuronic units converting theminto iduronic units and introducing O-sulfate groups to get a productwhich, once extracted industrially, has approximately double the numberof sulfate groups as regards carboxyl ones per disaccharide unit. Theseenzymatic modifications lead to, besides, the formation of thepentasaccharide region of a bond to antithrombin III (ATIII), calledactive pentasaccharide, which is the structure necessary for the highaffinity bond of heparin to the ATIII and fundamental for anticoagulantand antithrombotic activity of the heparin itself. This pentasaccharide,present inside only some of the chains which form heparin, contains asulfated glucosamine unit in position 3 and a glucuronic acid spaced outbetween disaccharides containing iduronic acids.

In nature, the formation of the active pentasaccharide is made possibleby the epimerization reaction of the carboxyl of a part of theglucuronic units into iduronic units carried out by theglucuronyl-C5-epimerase (C5-epimerization) and by suitable sulfationwhich also leads to the introduction of a sulfate group onto thehydroxyl in position 3 of the glucosamine. More particularly, in naturethe formation of the active pentasaccharide is made possible by the factthat the C5-epimerization occurs in clusters, i.e. on portions ofchains, and extensively, which results in a product that contains moreiduronic units than glucuronic ones. Commercial heparin, in fact,contains approximately 70% of iduronic units and 30% of glucuronicunits. Alongside the main anticoagulant and antithrombotic activities,heparin also exercises antilipaemic, antiproliferative, antiviral,antitumorous and antimetastatic activities, but its use as a drug ishindered by the side effects due to the anticoagulant action which cancause bleeding.

PRIOR ART

It is known that the capsular K5 polysaccharide isolated fromEscherichia coli, described by Vann W. F. et al., in European Journal ofBiochemistry, 1981, 116, 359-364 (“Vann 1981”), consists of a chainmixture consisting of the repetitive disaccharide unitglucuronyl-β-1→4-N-acetyl glucosamine and therefore shows the samerepetitive sequence (A)

of the N-acetyl-heparosan precursor of heparin. The capsular K5polysaccharide, referred to hereafter as “K5 polysaccharide” or moresimply “K5”, was chemically modified by Lormeau et al. as described inU.S. Pat. No. 5,550,116 and by Casu et al. as described in CarbohydrateResearch, 1994, 263, 271-284. K5-O-sulfates having antitumorous,antimetastatic, antiviral, in particular anti-HIV activities aredescribed in EP 333243 and WO 98/34958. The K5 was also modifiedchemically and enzymatically in order to obtain products having the sametype of in vitro biological activity on coagulation as that of heparinas extracted from animal organs (extractive heparin).

The attainment of the products having an activity on coagulation of thesame type as that of extractive heparin occurs by processes whichimitate that occurring in nature and envisage the entire key step ofC5-epimerization with D-glucuronyl C5 epimerase.

The processes described in IT 1230785, WO 92/17507, WO 96/14425 and WO97/43317 utilize K5 as the starting material. K5 originating fromfermentation is subjected to N-deacetylation followed by N-sulfation andon the K5-N-sulfate thus obtained C5-epimerization with C5-epimerase insolution is performed, obtained either by chromatography of a solutionof microsomal enzymes from mouse mastocytoma (IT 1230 785) or frombovine liver (WO 92/17507, WO 96/14425 and WO 97/43317).

The D-glucuronyl C5 epimerase from bovine liver was purified byCampbell, P. et al. in J. Biol. Chem., 1994, 269/43, 26953-26958(“Campbell 1994”) who also supplied its composition in amino acids anddescribed its use in solution for the transformation of a K5-N-sulfateinto the corresponding 30% epimerized product, demonstrating theformation of iduronic acid by HPLC method followed by total nitrousdepolymerization to disaccharide.

The document WO 98/48006 describes the DNA sequence which codes for theD-glucuronyl C5 epimerase and a recombinant D-glucuronyl C5 epimerase,obtained from a recombinant expression vector containing said DNA,afterwards purified by Campbell et al. as shown by Jin-Ping L. et al. inJ. Biol. Chem. 2001, 276, 20069-20077 (“Jin-Ping 2001”).

The complete C5-epimerase sequence was described by Crawford B. E. etal. in J. Biol. Chem., 2001, 276(24), 21538-21543 (Crawford 2001).

The document WO 01/72848 describes a method for the preparation ofN-deacetylated N-sulfate derivatives of K5 polysaccharide, at least 40%epimerized of iduronic acid as regards the total of the uronic acids,having a molecular weight from 2,000 to 30,000, containing from 25 to50% of high affinity chains for ATIII and having an anticoagulant andantithrombotic activity expressed as HCII/antiXa ratio from 1.5 to 4.Said document describes the oversulfation of a K5-N-sulfate, 40-60%epimerized and shows that the product obtained, whose ¹³C-RMN isillustrated, has a sulfate group content per disaccharide unit of 2-3.5.Repeating the aforesaid oversulfation in the conditions described andexamining the ¹³C-RMN it was ascertained that the product obtained isactually a free amine whose 6-O-sulfate content is 80-95%, that of3-O-sulfate on the amino sugar is 30%, but whose sulfation degree is3.2. It was also observed that in the conditions of oversulfationdescribed in WO 01/72848 a degree of sulfation higher than 3.2 was notobtained. The document U.S. 2002/0062019 describes a process for thepreparation of epiK5-N,O-sulfates, active in the control of coagulation,having a degree of sulfation from 2.3 to 2.9 and a molecular weight from2,000 to 30,000, or from 4,000 to 8,000, or from 18,000 to 30,000. Theaforesaid process involves the steps: (p-a) an N-deacetylation of K5polysaccharide and an N-sulfation of the resulting K5-amine, (p-b) anepimerization of K5-N-sulfate, (p-c) an O-oversulfation ofepiK5-N-sulfate, (p-d) a partial O-desulfation, (p-e) a selective6-O-sulfation, (p-f) an N-sulfation of the product thus obtained, anyproduct obtained upon termination of one of the steps (p-b)-(p-f) ableto be subjected to depolymerization. Said document describes anepiK5-N,O-sulfate having a molecular weight of 7,400, obtained by theaforesaid steps (p-a)-(p-f) followed by a nitrous depolymerization atthe end of step (p-f), with a degree of sulfation from 2.3 to 2.9.

The same document also describes a moiety of K5 with a molecular weightof approximately 5,000 which can also be subjected to steps (p-a)-(p-f).

In order to standardize the terminology and render the text morecomprehensible, in the present description conventional terms orexpressions will be used, in the singular or plural. In particular:

-   -   by “K5” or “K5 polysaccharide” is meant the capsular        polysaccharide from Escherichia coli obtained by fermentation,        i.e. a chain mixture consisting of disaccharide units (A)        optionally containing a double bond at the non-reducing end as        shown above, in any case, prepared and purified according to the        methods described in literature, in particular according to Vann        1981, according to Manzoni M. et al., Journal of Bioactive        Compatible Polymers, 1996, 11, 301-311 (“Manzoni 1996”) or        according to the method described in WO 01/72848 and in WO        02/068447; it is obvious for a person skilled in the art that        what is shown hereafter can be applied to any N-acetylheparosan;    -   by “C5-epimerase” is meant the D-glucuronyl C-5 epimerase,        extractive or recombinant, in any case prepared, isolated and        purified, in particular as described in Campbell 1994, in WO        98/48006, in Jin-Ping L. et al. in J. Biol. Chem. 2001, 276,        20069-20077 (Jin-Ping 2001”) or in Crawford 2001;    -   by K5-amine is meant at least 95% N-deacetylated K5, but in        which N-acetyl groups are undetectable with a normal NMR        apparatus;    -   by “K5-N-sulfate” is meant at least 95% N-deacetylated and        N-sulfate K5, normally 100%, since N-acetyl groups are        undetectable with a normal NMR apparatus, as described        hereafter;    -   by “epiK5” is meant the K5 and its derivatives in which 20-60%        of the glucuronic units is C5-epimerized to iduronic units    -   by “epiK5-N-sulfate” is meant K5-N-sulfate in which 20-60% of        the glucuronic units is C5-epimerized to iduronic units;    -   by “epiK5-amine-O-oversulfate” is meant an epiK5-amine-O-sulfate        with a sulfation degree of at least 3.4;    -   by “epiK5-N,O-oversulfate” is meant an epiK5-amine-O-sulfated        completely N-sulfated with a sulfation degree of at least 4;

In addition:

-   -   the conventional terms and expressions herein defined above        refer to a K5 as isolated after fermentation, generally with a        molecular weight distribution from approximately 1,500 to        approximately 50,000 with a mean molecular weight of        10,000-25,000, advantageously of 15,000-25,000;    -   the conventional terms and expressions herein defined above,        when preceded by the acronym “LMW” (low molecular weight), for        example LMW-K5-N-sulfate, LMW-epiK5-N-sulfate, indicate low        molecular weight products, obtained by fractionation or by        depolymerization of K5-N-sulfate and consisting of or derived        from K5-N-sulfates having a mean molecular weight from        approximately 1,500 to approximately 12,000, calculated on a        100% N-sulfated product;    -   the conventional terms and expressions as herein defined above,        when followed by “-derivative” indicate as a whole both the        derivatives from native K5 and those of low molecular weight;    -   by the term “approximately”, referring to the molecular weight,        is meant the molecular weight measured by viscometry±the        theoretical weight of a disaccharide unit, including the weight        of the sodium, calculated as 461 in the case of an        epiK5-N-sulfate-derivative and 806 in the case of an        epiK5-N,O-oversulfated-derivative with a sulfation degree of        4.26;    -   by the expression “preponderant species”, is meant the compound        which, in the mixture constituting the LMW-epiK5-N-sulfate, the        LMW-epiK5-amine-O-oversulfate or the LMW-epiK5-N,O-oversulfate,        is the most represented type, determined by the peak of the        curve of the molecular weight measured by HPLC;    -   unless otherwise specifically stated, by “degree of sulfation”        is meant the SO₃ ⁻ /COO⁻ ratio, expressible also as the number        of sulfate groups per disaccharide unit, measured with the        conductometric method described by Casu B. et al. in        Carbohydrate Research, 1975, 39, 168-176 (Casu 1975), the same        utilized in WO 01/72848;    -   by “conditions of O-oversulfation” is meant an extreme        O-sulfation performed, for example, according to the Method C        described by B. Casu et al. in Carbohydrate Research, 1994, 263,        271-284 (Casu 1994);    -   by the term “alkyl” is meant a linear or branched alkyl, whereas        “tetrabutylammonium” denotes the tetra-n-butylammonium group.

SUMMARY OF THE INVENTION

It has now surprisingly been found that, unlike that which occurs withthe processes described in IT 1230785, WO 92/17507, WO 96/14425, WO97/43317, WO 01/72848 and U.S. 2002/0062019, starting with anepiK5-N-sulfate it is possible to obtain an epiK5-amine-O-oversulfatewith a greater degree of sulfation than every otherepiK5-amine-O-sulfate described in literature, for example in WO01/72848, by preparing the salt with tertiary or quaternary organic baseof said epiK5-N-sulfate taking care to let the reaction mixture to standfor a time period of 30-60 minutes maintaining the pH at approximately 7with the same organic base and then treating the salt obtained with anO-sulfation reagent in the conditions of O-oversulfation.

Subjecting the epiK5-amine-O-oversulfates thus obtained to N-sulfation,new epiK5-N,O-oversulfates are obtained which, unlike the productsdescribed in IT 1230785, WO 92/17507, WO 96/14425, WO 97/43317, WO01/72848 and U.S. 2002/0062019, are free of activity on coagulation anduseful for the preparation of medicines, particularly pharmaceuticalcompositions of antiangiogenetic and antiviral activity or of cosmeticcompositions.

By depolymerization with nitrous acid of said epiK5-N,O-oversulfates newLMW-epiK5-N,O-oversulfates are obtained, free of activity oncoagulation, and with antiangiogenetic and antiviral activity.

In preparing N,O-sulfate N-deacetylated derivatives of K5polysaccharide, at least 40% epimerized of iduronic acid as regards thetotal of the uronic acids and having low molecular weight according tothe method described in WO 01/72848, it was ascertained that thedepolymerization of the product of high molecular weight obtained at theend of the final N-sulfation step of the process can give varyingresults since it generally produces some depolymerized products showingmuch lower activity, than that of high molecular weight products fromwhich they arise, on all the coagulation parameters. It is assumed thistakes place because degradation with nitrous acid is influenced by thepresence of the sulfate groups. In particular the sulfates in position 3of the glucosamine result in heterogenous products, as described byNagasawa et al. in Thrombosis Research, 1992, 65, 463-467 (Nagasawa1992).

It has now been found that subjecting an epiK5-N-sulfate to nitrousdepolymerization in which the iduronic acid content as regards the totalof uronic acids is 20-60%, advantageously of 40-60%, preferably around50%, LMW-epiK5-N-sulfates are obtained which constitute new effectiveintermediates for the preparation of LMW-epiK5-N,O-oversulfates having ahigh degree of activity on different biological parameters, with orwithout activity on coagulation parameters. In particular, it was foundthat it is possible to depolymerize an epiK5-N-sulfate so as to obtainnew LMW-epiK5-N-sulfates of mean molecular weight from approximately2,000 to approximately 4,000, more particularly specificLMW-epiK5-N-sulfates consisting of mixtures in which the predominantcompound is a decasaccharide or a dodecasaccharide or atetradecasaccharide. Also these LMW-epiK5-N-sulfates, otherwiseunobtainable, have interesting biological properties and are usefulintermediates for the preparation of LMW-epiK5-N,O-oversulfated ofantiviral and/or antiangiogenetic activity and surprisingly free ofactivity on coagulation.

By subjecting a LMW-epiK5-N-sulfate to the aforesaid method ofsalification with a tertiary or quaternary organic base, taking care tolet the reaction mixture to stand for a time period of 30-60 minutesmaintaining the pH at approximately 7 with the same organic base andthen treating the salt obtained with an O-sulfation reagent in theconditions of O-oversulfation, new LMW-epiK5-amine-O-oversulfates areobtained. By subjecting the LMW-epiK5-amine-O-oversulfate toN-sulfation, new N-sulfated and O-oversulfated derivatives(LMW-epiK5-N,O-oversulfates) are obtained, surprisingly free of activityon coagulation and of antiviral and/or antiangiogenetic activity, usefulfor the preparation of pharmaceutical or cosmetic compositions.

These LMW-epiK5-N-sulfates are obtained starting with a K5-N-sulfatewith an epimerization reaction with an isolated and purified recombinantC5-epimerase, immobilized on a solid support, at a temperature ofapproximately 30° C. and at a pH of approximately 7 for 12-24 hours inthe presence of a bivalent cation selected among calcium, magnesium,barium and manganese and a subsequent nitrous depolymerization reactionof the epimerized product thus obtained, or vice versa.

Surprisingly, from observations made on the course of the epimerizationreaction in the aforesaid conditions, it is possible to assume that,contrary to that occurring in nature in the biosynthesis of heparin,ordinary and not “cluster” type C5-epimerization of the substrate occursevery 2 glucuronic acid units which leads toepi-K5-N-sulfate-derivatives characterized by a repetitivetetrasaccharide unit consisting of two glucosamine units separated by aglucuronic unit and followed by an iduronic unit or vice versa.

DETAILED DESCRIPTION

Thus, according to one of its aspects, the present invention provides aprocess for the preparation of epiK5-N,O-oversulfate-derivatives,characterized in that

-   (a) a K5-N-sulfate-derivative, in acidic form, is treated with a    tertiary or quaternary organic base, letting the reaction mixture to    stand for a time period of 30-60 minutes, maintaining the pH of the    solution at a value of 7 by addition of the same tertiary or    quaternary organic base, and its salt is isolated with said organic    base;-   (b) said salt of organic base of said epiK5-N-sulfate-derivative is    treated with an O-sulfation reagent in the conditions of    O-oversulfation;-   (c) the epi-K5-amine-O-oversulfate-derivative thus obtained is    treated with a reagent of N-sulfation and the    epiK5-N,O-oversulfate-derivative is isolated.

Generally, the epiK5-N,O-oversulfate-derivative is isolated in sodiumsalt form and optionally said sodium salt is transformed into anotherchemically or pharmaceutically acceptable salt.

In this context, the term “chemically acceptable” refers to a cationusable in chemical synthesis, such as the sodium, ammonium,(C₁-C₄)tetraalkylammonium ion, or for the purification of the product,whereas “pharmaceutically acceptable” is self-explanatory.

Advantageous cations are those derived from alkaline metals,alkaline-earth metals, ammonium, (C₁-C₄)tetraalkylammonium, aluminum andzinc. Preferred cations are the sodium, calcium and tetrabutylammoniumions.

According to an advantageous manner of procedure, step (a) is carriedout by passing a solution of the sodium salt ofepiK5N-sulfate-derivative, i.e. of K5 polysaccharide, previouslyN-deacetylated, N-sulfated, normally 100%, and 20-60% C5-epimerized andoptionally depolymerized with nitrous acid, having a mean molecularweight from approximately 1,000 to approximately 25,000, advantageouslyfrom approximately 1,500 to approximately 25,000, through an acid ionicexchange resin, for example of the type IR-120H⁺, collecting the eluatealso including the washing water of the resin and neutralizing theeluate with tertiary or quaternary organic base, preferably with anaqueous solution of tetrabutylammonium hydroxide. The solution is let tostand for 1 hour, maintaining its pH at 7 by addition of the same baseand the salt thus obtained is isolated by lyophilization.

In step (b), the O-oversulfation occurs by using an excess ofO-sulfating agent and working at a temperature from 20 to 70° C. for atime period of up to 24 hours in an aprotic polar solvent.

Advantageously, the salt with a tertiary or quaternary organic base ofthe epiK5-N-sulfate-derivative, i.e. of K5 polysaccharide, previouslyN-deacetylated, N-sulfated preferably 100%, and 20-60% C5-epimerized andoptionally depolymerized with nitrous acid, having a mean molecularweight from approximately 1,000 to approximately 25,000, advantageouslyfrom approximately 1,500 to approximately 25,000 as isolated in step(a), is dissolved in dimethylformamide and treated with 2-10 moles of anO-sulfation reagent for every free hydroxyl at a temperature of 40-60°C. for 10-20 hours. As an O-sulfation reagent is advantageously used thepyridine.SO₃ adduct in a quantity of 2.5-5 moles, preferably 2.5-4 molesper free hydroxyl per disaccharide and the reaction is advantageouslycarried out at 50-60° C., preferably at 55° C., overnight. The productobtained upon termination of the reaction is isolated by addition of0.1-1 volume of water and neutralization, preferably with sodiumhydroxide, precipitation with a saturated sodium chloride solution inacetone, filtration and possible ultrafiltration.

The product thus obtained is generally the sodium salt of anepiK5-amine-O-oversulfate-derivative having an iduronic acid content of20-60% of the total of the uronic acids, having a mean molecular weightfrom approximately 3,500 to approximately 40,000, advantageously fromapproximately 4,500 to approximately 40,000 and a sulfation degree of atleast 3.4, advantageously of at least 3.5, more advantageously from 3.55to 4, preferably from 3.55 to 3.8. The sodium salt thus obtained can beconverted into another salt. By way of example an exchange with thecalcium ion can be performed working with ultrafiltration membranes.

In step (c), the epiK5-amine-O-oversulfated-derivative with a very highdegree of sulfation is N-sulfated using the known N-sulfation methods inliterature.

In practice, the N-sulfation is performed by treating an aqueoussolution containing the epiK5-amine-O-oversulfated-derivativeoriginating from step (b) with sodium carbonate and an agent ofN-sulfation, for example a (C₁-C₄)trialkylamine.SO₃ or pyridine.SO₃adduct, maintaining the mixture at 30-50° C. for 8-24 hours andisolating the desired epiK5-N,O-oversulfate-derivative, for example bydiafiltration. Optionally the step of N-sulfation is repeated untilobtaining more than 95% substitution, preferably complete.

The new epiK5-N,O-oversulfate-derivatives thus obtained are generally intheir sodium salt form. Said sodium salt can be converted into anotherchemically or pharmaceutically acceptable salt. Particularlyadvantageous salts are those of alkaline metals, alkaline-earth metals,of ammonium, (C₁-C₄)tetraalkylammonium, aluminum and zinc. Preferred arethe salts of sodium, calcium and tetrabutylammonium.

The starting epiK5-N-sulfates subjected to step (a) of the process ofthe present invention are derived from a K5 polysaccharide, previouslyN-deacetylated, N-sulfated virtually 100%, and 20-60% C5-epimerized,advantageously 40-60%, and optionally depolymerized with nitrous acid,having a mean molecular weight from approximately 1,000 to approximately25,000, advantageously from approximately 1,500 to approximately 25,000.Preferably, said starting material is an epi-K5-N-sulfate having a meanmolecular weight between 10,000 and 25,000 or a LMW-epiK5-N-sulfatehaving a mean molecular weight from approximately 1,000 to approximately12,000, advantageously from approximately 1,000 to approximately 10,000,preferably between approximately 1,500 and approximately 8,000.

The epiK5-N-sulfates, prepared by C5-epimerization of K5-N-sulfates, arewell known in literature and widely described for example in WO92/17507, WO 01/72848, WO 98/14425, WO 97/43317 or U.S. 2002/0062019.Their preparation by C-5 epimerization of the glucuronic unit ofK5-N-sulfate with a D-glucuronyl C5 epimerase was described in documentscited herein above.

A LMWepiK5-N-sulfate having an iduronic unit content of approximately20%, obtained by N-deacetylation, N-sulfation and C5-epimerization of amoiety of K5 having a mean molecular weight of 5,000 is described in WO92/17507. However such LMW-K5-N-sulfate contains a considerable quantityof acetyl groups.

An epiK5-N-sulfate with an iduronic acid content of 40-60%, particularlyadvantageous as a starting material, is that obtained by epimerizationof a K5-N-sulfate virtually free of acetyl groups, in turn prepared fromparticularly pure K5, in particular not containing lipophilicsubstances, described in WO 02/068477. According to a preferentialmanner of procedure, by epimerization a K5-N-sulfate is used obtainedfrom a K5 free of lipophilic substances like that described in WO02/068477 and the C5 epimerization is performed with a D-glucuronylC5-epimerase that is isolated, purified and immobilized on a solidsupport, at a pH of approximately 7, at a temperature of approximately30° C. and for a time period of 12-24 hours in the presence of at leastone bivalent ion selected among calcium, magnesium, barium andmanganese.

The LMW-epiK5-N-sulfates having a higher content of iduronic units, inparticular 40-60%, preferably 50-55%, are new, particularly advantageousproducts as starting materials in the preparation ofLMW-epiK5-N,O-oversulfate-derivatives.

The LMW-epiK5-N-sulfates as shown above are prepared by a processcharacterized in that a K5-N-sulfate is subjected, in any one order,

-   (i) to C5-epimerization with a D-glucuronyl C5-epimerase that is    isolated, purified and in solution or immobilized on a solid    support, at a pH of approximately 7, at a temperature of    approximately 30° C. and for a time period of 12-24 hours in the    presence of at least one bivalent ion selected among calcium,    magnesium, barium and manganese; and-   (ii) to a nitrous depolymerization optionally followed by reduction,    normally with sodium borohydride.

The expression “in any order” shows that the process can beindifferently carried out both in the direction (i)-(ii), i.e. in thesequence shown above, as well as in reverse direction, i.e. also in thedirection (ii)-(i), subjecting the K5-N-sulfate at first to the nitrousdepolymerization reaction, optionally followed by reduction with sodiumborohydride, and afterwards to C5-epimerization in the aforesaidconditions. The preferred order is in the direction (i)→(ii). Thesequence (ii)-(i) is preferably utilized starting with LMW-K5-N-sulfateshaving a mean molecular weight of more than 4,000, preferably startingwith approximately 6,000. For example, one can determine the amount ofsodium nitrite which, starting with 1 g of epiK5-N-sulfate, allows theattainment of a LMW-epiK5-N-sulfate with a molecular weight of more than4,000, in particular of at least 6,000, so as to obtain usefulintermediates for the preparation of LMWepiK5-N,O-oversulfates. In fact,in this case, in step (ii) the percentage of optimum epimerization isobtained.

According to a preferential aspect of the invention, the C5-epimerase isimmobilized on an inert solid support.

The C5-epimerase, preferably recombinant, isolated and purified forexample according to Campbell 1994, WO 98/48006, Jin-Ping 2001 orCrawford 2001, is immobilized on an inert support in the presence of thesubstrate, i.e. in the presence of starting K5-N-sulfate-derivative orin the presence of LMW-K5-N-sulfate, advantageously having a meanmolecular weight of more than 4,000, preferably of at least 6,000. Theimmobilization is performed according to conventional methods, forexample as described in WO 01/72848.

The C-5epimerization reaction is carried out by recirculating 20-1,000ml of a 25 mM HEPES solution at a pH of approximately 0.7 containing0.001-10 g of substrate (K5-N-sulfate or LMW-K5-N-sulfate, preferablywith a molecular weight of more than 4,000, in particular of at least6,000) and a cation selected among calcium, magnesium, barium andmanganese at a concentration of between 10 and 60 mM through a columncontaining from 1.2×10⁷ to 3×10¹¹ cpm of the immobilized enzyme,maintaining the pH at approximately 7 at approximately 30° C., at a flowof 30-220 ml/hour for a time period of 12-24 hours, advantageously 15-24hours.

Preferably said solution is recirculated at a flow of approximately 200ml/hour overnight (15-20 hours). The product obtained is purified andseparated according to known methods, for example by ultrafiltration andprecipitation with ethanol. The product thus obtained is eitherconsisting of epiK5-N-sulfate (and in such case is dissolved in waterand subjected to depolymerization) or LMW-epiK5-N-sulfate (in such caseit constitutes the end product). The percentage of epimerization, inpractice the amount of iduronic units as regards the glucuronic ones, iscalculated using ¹H-RMN according to the method described in WO 96/4425.

The nitrous depolymerization reaction is carried out according to knownmethods by the depolymerization of heparin, for example according to themethod described in EP 37319, in WO 82/03627 or according to the methodby depolymerization of a K5-N-sulfate described in EP 544592, butstarting with a K5-N-sulfate or an epiK5-N-sulfate containing from 0 tono more than 10%, preferably no more than 5%, of acetyl groups.Preferably, the depolymerization, performed with sodium nitrite andhydrochloric acid on an epiK5-N-sulfate virtually free of acetyl groups,is followed by in situ reduction with sodium borohydride.

In practice, a cold aqueous solution of epiK5-N-sulfate is brought toacid pH (approximately 2) with hydrochloric acid and, still cold,treated with sodium nitrite maintaining the temperature (approximately4° C.) and the pH (approximately 2) constant and, upon termination ofdepolymerization (approximately 15-30 minutes) the solution isneutralized with sodium hydroxide and treated, still at approximately 4°C., with an aqueous solution of sodium borohydride. Upon termination ofthe reduction (approximately 4 hours) the excess sodium borohydride isdestroyed with hydrochloric acid, the solution is neutralized withsodium hydroxide and the depolymerized (and reduced) product is isolatedaccording to known methods, for example by straightforward precipitationwith ethanol or acetone. The product obtained upon termination ofdepolymerization can be either a LMW-epiK5-N-sulfate (in such case itconstitutes the end product) or a LMW-K5-N-sulfate (and in such case isdirectly subjected to C5-epimerization as herein shown above, afterisolation or also in solution without being previously isolated), inparticular when it has a mean molecular weight of more than 4,000,preferably of at least 6,000, or is utilized to prepare antiangiogeneticand antiviral activity LMW-K5-N,O-oversulfated. By appropriatelycontrolling the depolymerization reaction, in particular using differentamounts of sodium nitrite/hydrochloric acid, are obtainedLMW-K5-N-sulfates or LMW-epiK5-N-sulfates having a mean molecular weightin the entire interval from approximately 1,500 to approximately 12,000,advantageously from approximately 1,500 to approximately 10,000,preferably from approximately 1,500 to approximately 7,500, calculatedat the ¹³C-RMN spectrum through the integration of the signal attributedto the C2 of 2,5-anhydromannitol with that of the anomeric carbon of theglucosamine inside the polysaccharide chain. According to a generalmanner of procedure, starting for example with 1 g of epiK5-N-sulfate,the starting product is dissolved in 100-200 ml of deionized water andthermostated at 4° C. Then an amount of sodium nitrite is added so as toobtain the desired mean molecular weight, for example from approximately2,000 to approximately 4,000. Therefore, starting with anepiK5-N-sulfate having a molecular weight of 20,000 measured with theHPLC method equipped with a BioRad BioSil 250 column and using a heparinstandard of known molecular weight, will require the addition of 330 to480 mg of sodium nitrite dissolved in a 0.2% aqueous solution. Thesolution containing the epiK5-N-sulfate and the sodium nitrite, kept at4° C., is brought to pH 2 through the addition of 0.1 N HCl cooled to 4°C. It is left to react under slow agitation for 20-40 minutes, then isneutralized with 0.1 N NaOH. The product obtained is brought to roomtemperature and treated with reducing agent such as for example sodiumborohydride (250-500 mg dissolved in 50-100 ml of water) and left toreact for 4-8 hours. The excess sodium borohydride is eliminatedbringing the pH to 5-5.5 with 0.1 N HCl and let to stand for a further2-4 hours. In the end it is neutralized with 0.1 N NaOH and the productis recovered by precipitation with acetone or ethanol after havingconcentrated the product by evaporation at reduced pressure.

Similarly, the amount of sodium nitrite can be determined which,starting with 1 g of K5-N-sulfate or epiK5-N-sulfate, allows theattainment of a LMW-K5-N-sulfate or a LMW-epiK5-N-sulfate with a meanmolecular weight from approximately 4,000 to approximately 12,000,advantageously from approximately 4,000 to approximately 7,500, inparticular of 6,000-7,500.

The LMW-epiK5-N-sulfate thus obtained, with an iduronic acid contentfrom 20 to 60%, advantageously from 40 to 60%, preferably of 50-55% andvirtually free of NH₂ and N-acetyl groups, having a mean molecularweight from approximately 1,500 to approximately 12,000, advantageouslyfrom approximately 1,500 to approximately 10,000, preferably fromapproximately 1,500 to approximately 7,500 and their chemically orpharmaceutically acceptable salts constitute new products useful asparticularly interesting starting materials in the preparation ofLMW-epiK5-N,O-oversulfates, but also themselves useful as activeingredients of pharmaceutical or cosmetic compositions and constitute anadditional aspect of the present invention.

Advantageously, the starting materials in the preparation of theepiK5-N,O-oversulfate-derivatives of the present invention areepiK5-N-sulfate-derivatives consisting of a chain mixture in which atleast 90% of said chains have the formula I

in which the uronic units are 20-60% consisting of iduronic acid, n is ainteger from 2 to 100, advantageously from 3 to 100 and thecorresponding cation is chemically or pharmaceutically acceptable. Moreadvantageously, said starting epiK5-N-sulfate-derivatives are consistingof a chain mixture in which at least 90% of said chains have the formulaI in which the uronic units are 40-60% consisting of iduronic acid, n isa integer from 2 to 100, advantageously from 3 to 100 and thecorresponding cation is chemically acceptable. Preferred startingmaterials are LMW-epiK5-N-sulfates as shown above, consisting of a chainmixture in which at least 90% of said chains have the formula I in whichthe uronic units are 20-60% comprised advantageously 40-60%, preferably50-55%, of iduronic acid, n is a integer from 2 to 20, advantageouslyfrom 3 to 15 and the corresponding cation is chemically acceptable.

In practice, said preferred LMW-epiK5-N-sulfates are consisting of achain mixture in which at least 90% of said chains have the formula I′

in which the uronic units are 20-60% comprised, advantageously 40-60%,preferably 50-55% of iduronic acid, q is a integer from 2 to 20,advantageously from 3 to 15, and the corresponding cation is chemicallyor pharmaceutically acceptable.

In this context, the term “chemically” refers to a cation usable inchemical synthesis, such as sodium, ammonium, (C₁-C₄)tetroalkylammoniumions, or for the purification of the product. Advantageous cations arethose derived from alkaline metals, alkaline-earth metals, ammonium,(C₁-C₄)tetraalkylammonium, aluminum and zinc. Preferred cations are thesodium, calcium and tetrabutylammonium ions.

Particularly interesting are the LMW-epiK5-N-sulfates consisting of achain mixture in which at least 90% of said chains have the formula I′herein above, obtained by nitrous depolymerization of the correspondingepiK5-N-sulfates shown above and subsequent possible reduction forexample with sodium borohydride. Among these, are preferred theLMW-epiK5-N-sulfates consisting of a chain mixture in which thepreponderant species has the formula I′a

in which the uronic units are 60-40% consisting of glucuronic acid and40% to 60% of iduronic acid, p is a integer from 4 to 8. The meanmolecular weight of these products is from approximately 2000 toapproximately 4000 and the corresponding cation is chemically orpharmaceutically acceptable.

The origin of these epiK5-N-sulfates from a step of nitrousdepolymerization involves, at the reducing end of the majority of thechains in said chain mixture, the presence of a 2,5-anhydromannose unitor, in the case of reduction with for example sodium borohydride, of2,5-anhydromannitol of structure (a)

in which X represents a formyl group or a hydroxymethyl group.Therefore, the reducing end of the majority (60-70% of the chains) isactually represented by the structure (b)

in which X is as defined above.

The presence of the structure (a) does not have any influence on thechemical characteristics of the epiK5-N-sulfates and their derivativessince any sulfations would lead to a possible introduction of one or twosulfate groups which would not however significantly move the sulfationdegree of the O-sulfated derivatives. It is however preferable that thenitrous depolymerization is followed by reduction for example withsodium borohydride since, according to the process of the presentinvention, said LMW-epiK5-N-sulfates are subjected to sulfation andacylation reactions whose influence, of the 2,5-anhydromannose radicalof structure (a), is unknown on the formyl group in which X representsformyl. Besides, the presence of structure (a) does not influence thebiological activity of the products, as demonstrated by Østergaard P. B.et al. in Thrombosis Research, 1987, 45, 739-749 (Østergaard 1987) forthe heparins of low molecular weight.

Particularly advantageous LMW-epiK5-N-sulfates according to the presentinvention are consisting of chain mixtures in which the preponderantspecies is a compound of formula I′b

in which X is formyl or, preferably, hydroxymethyl, m is 4, 5 or 6, thecorresponding cation is one chemically or pharmaceutically acceptableion and the glucuronic and iduronic units are present alternately,starting with a glucuronic or iduronic unit. In such case theglucuronic/iduronic ratio is from 45/55 to 55/45, i.e. approximately50/50.

The use of the C5-epimerase, preferably recombinant, preferablyimmobilized on a solid support in the conditions shown above thereforeallows not the “cluster” epimerization of K5-N-sulfate-derivatives intoepiK5-N-sulfate-derivatives as occurs in nature, but the ordinary type.

Thus, according to another of its aspects, the present inventionprovides the use of the isolated and purified C5-epimerase, for theconversion of a K5-N-sulfate-derivative into a correspondingepiK5-N-sulfate-derivative characterized by a repetitive tetrasaccharideunit consisting of two glucosamine units separated by a glucuronic unitand followed by an iduronic unit or separated by an iduronic unit andfollowed by a glucuronic unit.

Said epimerization occurs optimally if carried out on aK5-N-sulfate-derivative having a mean molecular weight of more than4,000, preferably from 6,000 to 7,500.

According to the present invention, the startingepiK5-N-sulfate-derivatives, preferably 100% N-sulfated (in particularthe epiK5-N-sulfate-derivatives consisting of chain mixtures in which atleast 90% of said chains have the formula I or I′ or in which thepreponderant species has the formula I′a or I′b where X ishydroxymethyl), are subjected to the aforesaid steps (a) and (b), upontermination of which are isolated the corresponding, newepiK5-amine-O-oversulfate-derivatives, in which the amine is notsubstituted, normally in sodium salt form, which can be transformed intoanother chemically or pharmaceutically acceptable salt. Particularlyadvantageous salts are those of alkaline metals, alkaline-earth metals,ammonium, (C₁-C₄)tetraalkylammonium, aluminum and zinc and, among these,the salts of sodium, calcium and tetrabutylammonium are preferred.

Thus, according to another of its aspects, the present invention refersto new epiK5-amine-O-oversulfate-derivatives and their chemically orpharmaceutically acceptable salts, obtainable by a process characterizedin that

-   (a) an epiK5-N-sulfate-derivative, in acidic form, is treated with a    tertiary or quaternary organic base, letting the reaction mixture to    stand for a time period of 30-60 minutes, maintaining the pH of the    solution at a value of 7 by addition of said tertiary or quaternary    organic base and its salt is isolated with said organic base;-   (b) said salt of organic base of said epiK5-N-sulfate-derivative is    treated with an O-sulfation reagent in the conditions of    O-oversulfation and the epiK5-amine-O-oversulfate-derivative is    isolated.

Using, as advantageous starting materials of step (a),epiK5-N-sulfate-derivatives consisting of a chain mixture in which atleast 90% of said chains has the aforesaid formula I, in which theuronic units are 20-60% consisting of iduronic acid, n is a integer from3 to 100 and the corresponding cation is chemically or pharmaceuticallyacceptable, at the end of step (b) anepiK5-amine-O-oversulfate-derivative is obtained consisting of a chainmixture in which at least 90% of said chains have the formula II

in which the uronic units are 20-60% consisting of iduronic acid, n is ainteger from 2 to 100, preferably from 3 to 100, R, R′ and R″ arehydrogen or SO₃ ⁻ , for a sulfation degree of at least 3.4,advantageously of at least 3.5, more advantageously from 3.55 to 4,preferably from 3.55 to 3.8 and the corresponding cation is chemicallyor pharmaceutically acceptable.

These epiK5-amine-O-oversulfate-derivatives with a very high degree ofsulfation are new products useful as intermediates in the preparation oftheir N-sulfate or N—(C₂-C₄)acylated derivatives basically free ofactivity on the coagulation parameters, but having other interestingpharmacological properties.

Advantageous epiK5-amine-O-oversulfate-derivatives with a very highdegree of sulfation are consisting of a chain mixture in which at least90% of said chains have the formula II in which the uronic units are40-60% consisting of iduronic acid, n is a integer from 2 to 100,preferably from 3 to 100, with a mean molecular weight fromapproximately 2,000 to approximately 40,000, advantageously fromapproximately 4,500 to approximately 40,000, R is at least 40%,preferably 50-80% SO₃ ⁻ , R′ and R″ are both SO₃ ⁻ or one is hydrogenand the other is 5-10% SO₃ ⁻ in monosulfate glucuronic acid and 10-15%SO₃ ⁻ in monosulfate iduronic acid, the degree of sulfation is more than3.4, advantageously of at least 3.5, more advantageously from 3.55 to 4,preferably from 3.55 to 3.8, and the corresponding cation is chemicallyor pharmaceutically acceptable.

Preferred epiK5-amine-O-oversulfate-derivatives with a very high degreeof sulfation are LMW-epiK5-amine-O-oversulfates consisting of a chainmixture in which at least 90% of said chains have the formula II inwhich the uronic units are comprised 40-60%, preferably 50-55%, ofiduronic acid, R is at least 40%, advantageously 50-80%, preferablyapproximately 65% SO₃ ⁻ , R′ and R″ are both SO₃ ⁻ or one is hydrogenand the other is 5-10% SO₃ ⁻ in glucuronic acid and 10-15% SO₃ ⁻ iniduronic acid, n is a integer from 2 to 20, advantageously from 3 to 15,with a mean molecular weight from approximately 4,000 to approximately8,000 and the corresponding cation is chemically or pharmaceuticallyacceptable.

In practice, said preferred LMW-epiK5-amine-O-oversulfates areconsisting of a chain mixture in which at least 90% of said chains havethe formula II′

in which the uronic units are 20-60% consisting of iduronic acid, q is ainteger from 2 to 20, advantageously from 3 to 15, R, R′ and R″ arehydrogen or SO₃ ⁻ , for a sulfation degree of at least 3.4,advantageously of at least 3.5, more advantageously from 3.55 to 4,preferably from 3.55 to 3.8, and the corresponding cation is onechemically or pharmaceutically acceptable ion.

Among these LMW-epiK5-amine-O-oversulfates are preferred thoseconsisting of a chain mixture in which the preponderant species has theformula II′a

in which the uronic units are 20-60% consisting of iduronic acid, p is ainteger from 4 to 8, R, R′ and R″ are as defined above, the degree ofsulfation is at least 3.4, advantageously of at least 3.5, moreadvantageously from 3.55 to 4, preferably from 3.55 to 3.8 and thecorresponding cation is chemically or pharmaceutically acceptable. Theorigin of the new LMW-epiK5-amine-O-oversulfates from LMW-epiK5-sulfatesobtained by nitrous depolymerization and subsequent reduction with, forexample, sodium borohydride, involves, at the reducing end of themajority of the chains in said chain mixture, the presence of a2,5-anhydromannitol sulfated unit of structure (a′)

in which R″ represents hydrogen or SO₃ ⁻ .

Thus, the reducing end of the majority of the chains in said chainmixture is represented by the structure (b′)

in which the uronic unit can be glucuronic or iduronic.

Among the aforesaid new LMW-epiK5-amine-O-oversulfates, are preferredthose consisting of mixtures in which the preponderant species is acompound of formula II′b

in which the uronic units are 40-60% consisting of iduronic acid, m is4, 5 or 6, R, R′ and R″ are hydrogen or SO₃ ⁻ , X″ is OH or OSO₃ ⁻ , fora sulfation degree of at least 3.4, advantageously of at least 3.5, moreadvantageously from 3.55 to 4, preferably from 3.55 to 3.8, the iduronicunits being present alternately, starting with a glucuronic or iduronicunit, and the corresponding cation is one chemically or pharmaceuticallyacceptable ion.

All these epiK5-amine-O-oversulfated-derivatives with a very high degreeof sulfation are new products which are useful intermediates for thepreparation of the new. N-substitutedepiK5-amine-O-oversulfated-derivatives and therefore constitute anadditional aspect of the present invention.

In particular, according to another of its aspects, the inventionconcerns the use of the aforesaid epiK5-amine-O-oversulfated-derivativeswith a very high degree of sulfation for the preparation of newN-substituted epiK5-amine-O-oversulfated-derivatives, in particularN-sulfated or N-acylated.

Upon termination of step (c) of the process of the present invention,consisting of an N-sulfation of theepiK5-amine-O-oversulfate-derivatives obtained at the end of step (b)(in particular the epiK5-amine-O-oversulfate-derivatives consisting ofchain mixtures in which at least 90% of said chains have the formula IIor II′ or in which the preponderant species has the formula II′a orII′b) epiK5-N,O-oversulfate-derivatives are obtained whose iduronic acidcontent is 20-60% of the total of the uronic acids and whose sulfationdegree is at least 4, preferably from 4 to 4.6.

Thus, according to another of its aspects, the present inventionprovides new N-deacetylated derivatives of K5 polysaccharide, O-sulfatedand N-sulfated, C5-epimerized to iduronic acid in at least 20% of thetotal of the uronic units, having a mean molecular weight fromapproximately 2,000 to approximately 45,000, a sulfation degree of atleast 4, said derivatives being basically inactive on the coagulationparameters.

Similarly to that stated above, said new derivatives are, as a whole,denoted by the general term “epiK5-N,O-oversulfate-derivatives”,independently of their molecular weight.

In particular, the mean molecular weight is between approximately 2,000to approximately 45,000 since said derivatives originate either from anepi-K5-N-sulfate obtained by N-deacetylation and N-sulfation of K5 byfermentation or by the nitrous depolymerization of the latter. Bycontrolling said nitrous depolymerization it is possible to obtain lowmolecular weight derivatives in virtually all the aforesaid interval.However, for use of the derivatives of the present invention aspharmaceutical or cosmetic products it is advantageous to prepare lowmolecular weight derivatives, with a mean molecular weight fromapproximately 2,000 to approximately 16,000, advantageously fromapproximately 3,500 to approximately 13,000 with a molecular weightdistribution of between approximately 1,000 and approximately 15,000,preferably from approximately 4,500 to approximately 9,000, with amolecular weight distribution from approximately 2,000 to approximately10,000, or of high molecular weight derivatives, originating from theunfractionated K5, with a mean molecular weight of between approximately20,000 and approximately 45,000, with a molecular weight distributionfrom approximately 2,000 to approximately 70,000.

In the epiK5-N,O-oversulfate-derivatives of the present invention thedegree of sulfation is very high, preferably from 4 to 4.6, the nitrogenof the glucosamine being virtually 100% sulfated. Besides, theepiK5-N,O-oversulfate-derivatives are 100% 6-O-sulfated and 50-80%3-O-sulfated in their glucosamine units, 5-10% 3-O-monosulfated inglucuronic units, 10-15% O-monosulfated in iduronic units and2.3-di-O-sulfated in the remaining uronic units, considering that thedegree of sulfation is at least 4.

Advantageous epiK5-N,O-oversulfate-derivatives according to the presentinvention are obtained through epiK5-amine-O-oversulfate-derivatives inturn prepared from epiK5-N-sulfate-derivatives consisting of a chainmixture in which at least 90% of said chains has the aforesaid formulaI, in which the uronic units are 20-60% consisting of iduronic acid, nis a integer from 2 to 100, advantageously from 3 to 100 and thecorresponding cation is chemically or pharmaceutically acceptable.

In such case, the new epiK5-N,O-oversulfate-derivatives consisting ofchain mixtures in which at least 90% of said chains have the formula III

in which the uronic units are 20-60% consisting of iduronic acid, n is ainteger from 2 to 100, preferably from 3 to 100, R, R′ and R″ arehydrogen or SO₃ ⁻ , Z is SO₃ ⁻ , the degree of sulfation is at least 4,preferably from 4 to 4.6 and the corresponding cation is chemically orpharmaceutically acceptable.

Said cations are advantageously those of alkaline metals, alkaline-earthmetals, ammonium, (C₁-C₄)tetraalkylammonium, aluminum and zinc and,among these, preferably the salts of sodium, calcium andtetrabutylammonium.

Among the aforesaid new epiK5-amine-N,O-oversulfate-derivatives, thoseconsisting of chain mixtures in which at least 90% of said chains hasthe aforesaid formula III in which R is SO₃ ⁻ in 50%-80%, preferably inapproximately 65% of said chains and the degree of sulfation is at least4, advantageously is from 4 to 4.6, preferably from 4 to 4.3.

Advantageous epiK5-N,O-oversulfate-derivatives with a very high degreeof sulfation are consisting of a chain mixture in which at least 90% ofsaid chains have the formula II, in which Z is SO₃ ⁻ , the uronic unitsare 40-60% consisting of iduronic acid, n is a integer from 2 to 100,preferably from 3 to 100, with a mean molecular weight fromapproximately 2,000 to approximately 45,000, advantageously fromapproximately 4,500 to approximately 45,000, R is at least 40%,preferably 50-80% SO₃ ⁻ , R′ and R″ are both SO₃ ⁻ or one is hydrogenand the other is 5-10% SO₃ ⁻ in monosulfate glucuronic acid and 10-15%SO₃ ⁻ in monosulfate iduronic acid, the degree of sulfation is at least4, from 4 to 4.6 and the corresponding cation is chemically orpharmaceutically acceptable.

Preferred N-substituted epiK5-amine-O-oversulfated-derivatives areLMW-epiK5-amine-O-oversulfated consisting of a chain mixture in which atleast 90% of said chains have the formula III in which the uronic unitsare 40-60% comprised, preferably 50-55%, of iduronic acid, R is at least40%, advantageously 50-80%, preferably approximately 65% SO₃ ⁻ , R′ andR″ are both SO₃ ⁻ or one is hydrogen and the other is 5-10% SO₃ ⁻ inglucuronic acid and 10-15% SO₃ ⁻ in iduronic acid, Z is 100% SO₃ ⁻ or(C₂-C₄)acyl, n is a integer from 2 to 20, preferably from 3 to 15, witha mean molecular weight from approximately 4,000 to approximately 8,500and the corresponding cation is chemically or pharmaceuticallyacceptable.

In practice, said preferred epiK5-N,O-sulfate-derivatives with a veryhigh degree of sulfation are consisting of a chain mixture in which atleast 90% of said chains have the formula III′

in which the uronic units are 20-60% consisting of iduronic acid, q is ainteger from 2 to 20, advantageously from 3 to 15, R, R′ and R″represent hydrogen or an SO₃ ⁻ group, Z is SO₃ ⁻ , for a sulfationdegree of at least 4, preferably from 4 to 4.6 and the correspondingcation is one chemically or pharmaceutically acceptable ion.

Particularly interesting are chain mixtures of formula III′ in which theuronic units are 40-60% comprised, preferably 50-55%, of iduronic acid,R is at least 40%, advantageously 50-80%, preferably approximately 65%SO₃ ⁻ , R′ and R″ are both SO₃ ⁻ or one is hydrogen and the other is5-10% SO₃ ⁻ in glucuronic acid and 10-15% SO₃ ⁻ in iduronic acid, n is ainteger from 2 to 20, advantageously from 3 to 15, with a mean molecularweight from approximately 2,000 to approximately 16,000, advantageouslyfrom approximately 3,500 to approximately 13,000, preferably fromapproximately 4,500 to approximately 9,000 and the corresponding cationis chemically or pharmaceutically acceptable. Among theseLMW-epiK5-N,O-oversulfates, are advantageous those consisting of a chainmixture in which the preponderant species has the formula III′a

in which the uronic units are 20-60% consisting of iduronic acid, p is ainteger from 4 to 8, Z is SO₃ ⁻ , R, R′ and R″ are hydrogen or SO₃ ⁻ ,for a sulfation degree of at least 4, preferably from 4 to 4.6 and thecorresponding cation is chemically or pharmaceutically acceptable. Theorigin of the new LMW-epiK5-N,O-oversulfates from LMW-epiK5-sulfatesobtained by nitrous depolymerization and subsequent reduction with, forexample, sodium borohydride involves, at the reducing end of themajority of the chains in said chain mixture, the presence of a sulfated2,5-anhydromannitol unit of structure (a′) as shown above, in which R″represents hydrogen or SO₃ ⁻ .

Thus, the reducing end of the majority of the chains in said chainmixture is represented by the structure (b″)

in which Z represents SO₃ ⁻ and the uronic unit can be glucuronic oriduronic.

Among the aforesaid new LMW-epiK5-N,O-oversulfates, are preferred thoseconsisting of mixtures in which the preponderant species is a compoundof formula III′b

in which R, R′ and R″ are hydrogen or SO₃ ⁻ , Z is SO₃ ⁻ , X″ is OH orOSO₃ ⁻ , m is 4, 5 or 6, for a sulfation degree of at least 4,preferably from 4 to 4.6, the uronic units are present alternately,starting with a glucuronic or iduronic unit, and the correspondingcation is one chemically or pharmaceutically acceptable ion. Saidcations are advantageously those of alkaline metals, alkaline-earthmetals, ammonium, (C₁-C₄)tetraalkylammonium, aluminum and zinc and,among these, preferably the ions of sodium, calcium andtetrabutylammonium.

If an epiK5 is used as a starting epiK5-derivative of the process of thepresent invention, i.e. a K5 polysaccharide, previously N-deacetylated,N-sulfated normally 100%, and 20-60% C5-epimerized and notdepolymerized, upon termination of step (c) an epiK5-N,O-oversulfate isisolated which can be subjected to nitrous depolymerization andpossible, subsequent reduction with, for example, sodium borohydride toobtain the corresponding LMW-epiK5-N,O-oversulfate having the samedegree of sulfation. In particular, LMW-epiK5-N,O-oversulfates areobtained consisting of a chain mixture in which at least 90% of saidchains have the formula III′ or III′a, in which the uronic units are20-60% consisting of iduronic acid, q, R, R′ R″ and Z have the meaningdefined above, for a sulfation degree of at least 4, preferably from 4to 4.6 and the corresponding cation is one chemically orpharmaceutically acceptable ion. In such case, the origin of theseLMW-epiK5-N,O-oversulfates from a depolymerization reaction andpossible, subsequent reduction with, for example, sodium borohydrideinvolves, at the reducing end of the majority of the chains in saidchain mixture, the presence of a 2,5-anhydromanno unit of structure (a″)

in which X is formyl or hydroxymethyl and R″ represents hydrogen or SO₃⁻ .

The new epiK5-N,O-oversulfate-derivatives, especially in their saltform, are highly anionic products able to capture the free radicals andare utilizable in the cosmetics industry as adjuvants against hair lossor to prepare “anti-ageing” creams and, in the pharmaceutical industry,as products for the treatment of dermatitis. Besides, theepiK5-N,O-oversulfate-derivatives of the present invention, inparticular the LMW-epiK5-N,O-oversulfates possess antiangiogenetic andantiviral activity and therefore constitute active ingredients for thepreparation of medicines.

Thus, according to one of its additional aspects, the present inventionprovides pharmaceutical compositions including, as one of their activeingredients, a pharmacologically active amount of anepiK5-N,O-oversulfate-derivative as shown above or of one of itspharmaceutically acceptable salts, in mixture with a pharmaceuticalexcipient.

In the pharmaceutical compositions of the present invention for oral,subcutaneous, intravenous, transdermal or topical administration, theactive ingredients are preferably administered in the form of dosageunits, in mixture with the classic pharmaceutical excipients orvehicles. The posology can vary widely depending on the age, weight, andthe health condition of the patient. This posology includes theadministration of a dose from 1 to 1000 mg, advantageously from 10 to750 mg, preferably 250 to 500 mg from one to three times a day byintravenous, subcutaneous, oral, transdermal or topical administration.The pharmaceutical compositions of the present invention are formulatedwith the classic excipients suitable for different ways ofadministration. Particularly advantageous are the formulations in theform of creams, ointments, liniments, gels, foams, balsams, vaginalpessaries, suppositories, solutions or suspensions suitable for localadministration.

Advantageously, the compositions of the present invention include, asone of its active ingredients, an epiK5-N,O-oversulfate-derivativeobtainable starting with an epiK5-derivative according to steps (a), (b)and (c) of the process described above, or starting with an epiK5 notdepolymerized, according to steps (a), (b) and (c) of the processdescribed above, with possible subsequent nitrous depolymerization afterstep (c), or one of its pharmaceutically acceptable salts, in mixturewith a pharmaceutical excipient. Advantageously, saidepiK5-N,O-oversulfate-derivative consists of a chain mixture in which atleast 90% of said chains have the formula III or III′ or in which thepreponderant species is a compound of formula III′a or III′b. Preferredactive ingredient is a LMW-epiK5-N,O-oversulfate having a sulfationdegree of at least 4, preferably from 4 to 4.6, advantageously having amean molecular weight from approximately 3,500 to approximately 11,000,more advantageously from approximately 3,500 to approximately 5,200 andbasically free of N-acetyl groups.

Finally, according to another of its aspects, the present inventionprovides a cosmetic composition including an effective amount of anepiK5-N,O-oversulfate-derivative or one of its pharmaceuticallyacceptable salts, in mixture with a cosmetic excipient.

A salt selected from the group consisting of salts of sodium, potassium,calcium, magnesium, aluminum and zinc of the epiK5-N,O-oversulfatederivatives, in particular those consisting of chain mixtures in whichat least 90% of said chains have the formula III or III′ or in which thepreponderant species has the formula III′a or III′b, constitutes aneffective active ingredient of the pharmaceutical or cosmeticcompositions of the present invention.

The following examples illustrate the invention without however limitingit.

Preparation I

Preparation of K5 Polysaccharide from Escherichia coli

At first fermentation is carried out in an Erlenmeyer flask using thefollowing medium: Fat-free soya meal 2 g/l K₂HPO₄ 9.7 g/l KH₂PO₄ 2 g/lMgCl₂ 0.11 g/l Sodium citrate 0.5 g/l Ammonium sulfate 1 g/l Glucose 2g/l

Spring water 1000 ml pH = 7.3

The medium is sterilized at 120° C. for 20 minutes. The glucose isprepared separately in solution form which is sterilized at 120° C. for30 minutes and added sterilely to the medium. The Erlenmeyer flask isinoculated with a suspension of E. coli cells Bi 8337/41 (O10:K5:H4)originating from a slant kept in Triptic soy agar, and incubated at 37°C. for 24 hours under controlled agitation (160 rpm, 6 cm stroke). Thebacterial growth is measured by counting the cells using a microscope.In a subsequent operation, a 14 l Chemap-Braun fermenter containing thesame medium as above, is 0.1% inoculated with the culture of theErlenmeyer flask as above and fermentation is performed by aeration of 1vvm, (vvm=volume of air per volume of liquid per minute) 400 rpmagitation and temperature of 37° C. for 18 hours. During fermentationare measured the pH, the oxygen, the glucose residue, K5 polysaccharideproduced and bacterial growth. At the end of fermentation thetemperature is brought to 80° C. for 10 minutes. The cells are separatedfrom the medium through centrifugation at 10,000 rpm and the supernatantis ultrafiltered using an SS 316 (MST) module fitted with PES membranewith nominal cut-off of 800 and 10,000 D to reduce the volume to ⅕. K5polysaccharide is then precipitated by addition of 4 volumes of acetoneat 4° C. and left to settle overnight at 4° C. Finally it is recoveredby centrifugation at 10,000 rpm for 20 minutes or filtration.Deproteinization of the solid obtained is carried out by using a type IIprotease from Aspergillus orizae in a buffer of 0.1 M NaCl and 0.15 MEDTA at pH 8 containing SDS (0.5% sodium dodecyl sulfate) (10 mg/l offiltrate) at 37° C. for 90 minutes. The solution obtained isultrafiltered on model SS 316 with membrane at a nominal cut-off of10,000 D with 2 extractions with 1M NaCl and washed with water untildisappearance of absorbance in the ultrafiltrate. K5 polysaccharide isthen precipitated with acetone and a yield of 850 mg per litre offermenter is obtained. The purity of the polysaccharide obtained ismeasured through the determination of the uronic acids (carbazolemethod), proton and carbon 13 NMR, UV and protein content. The purity ismore than 80%.

The polysaccharide obtained is composed of two moieties of differentmolecular weight, respectively 30,000 and 5,000 D as emerges from thedetermination by HPLC using a Pharmacia 75 HR column and a single moietywith a retention time of approximately 9 minutes using two seriatecolumns of Bio-sil SEC 250 (Bio Rad) and Na₂SO4 as mobile phase at roomtemperature and a flow of 0.5 ml/minute. The measurement is performedagainst a standard curve obtained with moieties of heparin of knownmolecular weight.

The ¹H-RMN spectrum of the purified K5 thus obtained shows differentsignals attributable to methyls of lipophilic substances.

Preparation II

Purification of K5

In 100 ml of a saturated aqueous solution of sodium chloride andthermostated at 4° C. is dissolved 1 g of K5 obtained at the end ofPREPARATION I and to the solution thus obtained are added 3 volumes ofcold isopropanol. The saline concentration of the solution is brought to3 M by addition of the calculated amount of a saturated sodium chloridesolution and the solution obtained is left in a cold environment(approximately 4° C.) overnight. The precipitate which forms isseparated by centrifugation at 10,000 rpm for 20 minutes and the purityof the product is checked by dialysis overnight and subsequentexamination of the ¹H-RMN spectrum, from which signals in the regionunder 1.5 ppm must be absent. Optionally, the operation of dissolutionin water saturated with NaCl and precipitation with isopropanol isrepeated. The precipitate is dissolved in water and ultrafiltered on aMiniplate Millipore membrane 10,000 D cut off until disappearance of thesalts. Thus a K5 having a purity of at least 99% is obtained from whose1H-RMN spectrum no traces of lipophilic impurities result in the regionunder 1.5 ppm.

Preparation III

Preparation of a K5-N-Sulfate

(i) N-Deacetylation

Ten grams of pure K5 polysaccharide prepared as described in PREPARATIONII are dissolved in 1000 ml of 2N sodium hydroxide and the solution thusprepared is left at 60° C. for 24 hours. The solution is brought to roomtemperature then to neutral pH (pH7) with 6N hydrochloric acid.

(ii) N-Sulfation

To the solution containing the deacetylated K5, kept at 40° C., areadded 16 g of sodium carbonate and afterwards and in 4 hours, 16 g ofpyridine.SO₃ ⁻ . At the end of the reaction, after 24 hours, thesolution is brought to room temperature, then to pH 6.5-7 with a 5%solution of hydrochloric acid. The product is purified from salts bydiafiltration using a 1,000 D helically wound membrane (prepscalecartridge—Millipore). The process is terminated when the conductivity ofthe permeate is less than 1000 μS, preferably less than 100 μS. Theintradialysis is reduced until a 10% concentration of the polysaccharideis obtained using the same in concentration dialysis system. Theconcentrated solution is dried by lyophilization. Upon ¹³C-RMN spectrumanalysis N-acetyl or NH₂ residues do not appear.

Preparation IV

LMW-K5-N-Sulfate

The product obtained as described in Example 1, steps (i) and (ii), ofWO 02/068477 is depolymerized by the degradation method with nitrousacid and subsequent reduction of the aldehyde which forms. One continuesby dissolving 1 g of K5-N-sulfate in 200 ml of distilled water andadding it with 480 mg of sodium nitrite dissolved in 240 ml of distilledwater. The solution is then brought to 4° C. and the pH to 2 with 0.1 NHCl and maintained for 30 minutes. At the end of the reaction thesolution is brought to pH 7 with 0.1 M NaOH and then to roomtemperature. The solution is then added with 450 mg. of NaBH₄ and leftto react for 4 hours. The excess NaBH₄ is eliminated with HCl bringingthe pH to 5-6. The product, neutralized with 0.1 M NaOH, is recovered byprecipitation with 3 volumes of acetone at 4° C., filtration withfiltering funnel and dried at 40° C. in a vacuum oven. 900 mg ofLMW-K5-N-sulfate are obtained with a mean molecular weight ofapproximately 2,000, consisting of a chain mixture in which thepreponderant species is a compound of formula I′b in which m is 4 andthe uronic units are those of glucuronic acid.

EXAMPLE 1

LMW-EpiK5-N-Sulfate. Sequence (i)→(ii)

(i) Epimerization to EpiK5-N-Sulfate

Ten grams of K5-N-sulfate obtained as described in Example 1, steps (i)and (ii), of WO 02/068477, from whose 1H-RMN spectrum, signalsconcerning acetyl groups or NH₂ do not appear, are dissolved in 600 mlof 25 mM HEPES buffer at pH 7, containing CaCl₂ at a concentration of 50mM and the solution thus obtained is made to recirculate through a 50 mlcolumn filled with Sepharose 4B resin containing 5 g of recombinantC5-epimerase (WO 96/14425) immobilized as described in Example 1 of WO01/72848. The reaction is carried out at 30° C. at pH 7 with a flow of200 ml/h for 24 hours. The product obtained is purified byultrafiltration and precipitation with ethanol. Thus an epiK5-N-sulfateis obtained whose iduronic acid content is 54%.

(ii) Depolymerization of EpiK5-N-Sulfate.

To a solution of 1 g of the product thus obtained, in 25 ml of distilledwater, are added 230 mg of sodium nitrite dissolved in 115 ml ofdistilled water. The solution is then brought to 4° C. and the pH to 2with 0.1 N HCl and maintained for 30 minutes. At the end of the reactionthe solution is brought to room temperature and the pH to 7 with 0.1 MNaOH. The solution is then added with 450 mg. of NaBH₄ and left to reactfor 4 hours. The product is recovered by precipitation with 3 volumes ofacetone at 4° C., filtration with filtering funnel and dried at 40° C.in a vacuum oven. 900 mg of LMW-epiK5-N-sulfate are obtained with aniduronic acid content of 54% and a molecular weight distribution from1,000 to 4,000, measured with HPLC method.

EXAMPLE 2

LMW-EpiK5-N-Sulfate. Sequence (ii)→-(i)

(ii) Depolymerization of K5-N-Sulfate

2 g of K5-N-sulfate, obtained as described in Example 1, steps (i) and(ii), of WO 02/068477, is depolymerized as described in PREPARATION I,using 100 mg of sodium nitrite and 300 mg of sodium borohydride. 1.8 gof LMW-K5-N-sulfate are obtained with a mean molecular weight of 5,000.

(i) Epimerization of LMW-K5-N-Sulfate

1 g of LMW-K5 N-sulfate obtained in step (ii) herein above is treated asdescribed in step (i) of the Example 1. An epimerized product isobtained with an iduronic acid/glucuronic acid ratio of 44/56 against aratio of 0/100 of the starting product, with a molecular weightdistribution from 2,000 to 10,000 and with a mean molecular weight of5,000 D. The yield, calculated by measuring the content of uronic acidsagainst a standard with the carbazole method (Bitter and Muir, Anal.Biochem. 1971, 39, 88-92) is 90%.

EXAMPLE 3

LMW-EpiK5-N-Sulfate. Sequence (i)→(ii)

(i) Epimerization of K5-N-Sulfate

A 2 g amount of K5 N-sulfate, obtained as described in Example 1, steps(i) and (ii), of WO 02/068477, is dissolved in 120 ml of 25 mM HEPESbuffer, pH 7, containing 50 mM CaCl₂. The solution obtained is made torecirculate through a 50 ml column filled with the resin containing theimmobilized enzyme obtained as described in WO 96/14425. This operationis carried out at 30° C. with a flow of 200 ml/h for 24 hours. Theproduct obtained is purified through ultrafiltration on a 1000 Dmembrane and passing over an IR 120H⁺ ionic exchange column,neutralizing the eluate with 1N NaOH. The sample is recovered byprecipitation with ethanol or acetone. An epimerized product is obtainedwith an iduronic acid/glucuronic acid ratio of 55/45 against a ratio of0/100 of the starting product. The percentage of epimerization wascalculated with ¹H-RMN according to the method described in WO 96/14425.The yield, calculated by measuring the content of uronic acids against astandard with the carbazole method (Bitter and Muir Anal. Biochem. 39,88-92-1971) is 90%.

(ii) Depolymerization of Epi-K5-N-Sulfate

One gram of product obtained in step (a) is depolymerized by thedegradation method with nitrous acid and subsequent reduction of thealdehyde which forms. In particular one continues by dissolving theproduct in 25 ml of distilled water and adding it with 230 mg of sodiumnitrite dissolved in 115 ml of distilled water. The solution is thenbrought to 4° C. and the pH to 2 with 0.1 N HCl and maintained for 30minutes. At the end of the reaction the solution is brought to roomtemperature and the pH to 7 with 0.1 M NaOH. The solution is then addedwith 450 mg. of NaBH₄ and left to react for 4 hours. The product isrecovered by precipitation with 3 volumes of acetone at 4° C.,filtration with filtering funnel and dried at 40° C. in a vacuum oven.900 mg of LMW-epiK5-N-sulfate are obtained with a molecular weightdistribution measured with HPLC method which ranges from 1,000 to 4,000and with a glucuronic unit content of 45% and an iduronic unit contentof 55%.

EXAMPLE 4

EpiK5-N,O-Oversulfate

(a) Tetrabutylammonium Salt of EpiK5-N-Sulfate

A solution in 40 ml of water of 400 mg of epiK5-N-sulfate, as obtainedat the end of step (i) of the Example 1, is thermostated at 4° C., thenpassed over IR 120⁺ ionic exchange resin preconditioned with water at 4°C. The eluate obtained, consisting of 100 ml of a solution at pH 1.94,is neutralized with a 15% solution of tetrabutylammonium hydroxide andleft at room temperature for one hour, maintaining the pH at 7 byaddition of 15% tetrabutylammonium hydroxide and finally is lyophilized.Thus 805 mg of tetrabutylammonium salt of epiK5-N-sulfate are obtained.

(b) Epi-K5-Amine-O-Oversulfate

A solution containing 805 mg of the salt thus obtained in 30 ml ofdimethylformamide is set at 55° C. and treated with 30 ml ofdimethylformamide containing 2.26 g of pyridine.SO₃ adduct. The reactionat 55° C. is continued overnight then 60 ml of water are added to themixture. After neutralization with 1 N NaOH, the product is precipitatedwith 3 volumes of acetone saturated with NaCl and set at 4° C.overnight. The precipitate is recovered by filtration on guch G4 andthen ultrafiltered with 1000 D TFF Millipore system and dried at reducedpressure. 550 mg of epi-K5-amine-O-oversulfated are obtained having acontent of iduronic acid of 54%, of glucosamine-6-O-sulfate of 100%, ofglucosamine 3-O-sulfate of 60%, of monosulfate glucuronic acid of 10%,of monosulfate iduronic acid of 15%, the rest of the uronic units beingdisulfated, with a sulfation degree of 3.55 measured with theconductometric method according to Casu et al. 1975.

(c) EpiK5-Amine-O-Oversulfated-N-Sulfate

To a solution of 250 mg of the epi-K5-amine-O-oversulfated obtained instep (b) in 15 ml of water are added 400 mg of sodium carbonate, then tothe mixture thus obtained are added 400 mg of pyridine.SO₃ adduct insolid form a little at a time in 4 hours. The reaction mixture is keptat 55° C. overnight, then is stopped bringing the pH to 7 with 0.1N HCl.After ultrafiltration on a 1000 D membrane are added 3 volumes ofacetone saturated with sodium chloride and the precipitate is recoveredby centrifugation at 5000 rpm for 5′. Thus 244 mg ofepiK5-N,O-oversulfate are obtained whose sulfation degree, measured withconductometric method according to Casu et al. 1975, is 4.25. By theanalysis of the ¹H-RMN spectrum it results that theepiK5-N,O-oversulfate thus obtained has an iduronic acid content of 54%,6-O-sulfate of 100%, N-sulfate of 100%, glucosamine 3-O-sulfate of 60%,monosulfate glucuronic acid of 10%, monosulfate iduronic acid of 15%,the rest of the uronic units being disulfated. From the ¹H-RMN spectrumis therefore calculated a sulfation degree of 4.35 which, consideringthe margins of error of the methods, corresponds to the sulfation degreeof epiK5-amine-O-oversulfated obtained upon termination of step (b),100% N-sulfated. It is therefore assumed that, beyond a certainpercentage of sulfate groups, the strong anionic nature of the productcan lead to an underestimation of the degree of sulfation determinedwith the conductometric method.

1-70. (canceled)
 71. A process for the preparation of anepiK5-N,O-oversulfate-derivative, which comprises (a) treating anepiK5-N-sulfate-derivative, in acidic form, with tertiary or quaternaryorganic base, letting the reaction mixture to stand for a time period of30-60 minutes at a pH of approximately 7 and its salt is isolated withsaid organic base; (b) treating said salt of organic base of saidepiK5-N-sulfate-derivative with an O-sulfation reagent in the conditionsof O-oversulfation; (c) treating a salt of tertiary or quaternaryorganic base of epiK5-amine-O-oversulfate-derivative thus obtained witha reagent of N-sulfation and isolating theepiK5-N,O-oversulfate-derivative thus obtained.
 72. Process according toclaim 71, wherein said epiK5-N,O-oversulfate-derivative is isolated insodium salt form and optionally transformed into another chemically orpharmaceutically acceptable salt.
 73. Process according to claim 71,wherein in step (a) tetrabutylammonium hydroxide is used as an organicbase.
 74. Process according to claim 71, wherein in step (b) theO-oversulfation is carried out in dimethylformamide using 2-4 moles ofO-sulfation reagent per available OH per disaccharide at a temperatureof 40-60° C. for 15-20 hours.
 75. Process according to claim 71, whereinan epiK5-N-sulfate-derivative is used as starting material having a meanmolecular weight from approximately 1,000 to approximately 25,000. 76.Process according to claim 75, characterized in that said startingepiK5-N-sulfate-derivative is 40-60% C5-epimerized.
 77. Processaccording to claim 71, wherein said starting epiK5-N-sulfate-derivativehas a mean molecular weight from approximately 1,500 to approximately25,000.
 78. Process according to claim 77, startingepiK5-N-sulfate-derivative has a mean molecular weight between 10,000and 25,000.
 79. Process according to claim 71, wherein said startingmaterial has a mean molecular weight from approximately 1,000 toapproximately 12,000.
 80. Process according to claim 79, wherein saidstarting material has a mean molecular weight from approximately 1,500to approximately 8,000.
 81. Process according to claim 71, wherein anepiK5-N-sulfate-derivative is used as starting material consisting of achain mixture in which at least 90% of said chains have the formula I

in which the uronic units are 20-60% consisting of iduronic acid, n isan integer from 2 to 100 and the corresponding cation is chemically orpharmaceutically acceptable.
 82. Process according to claim 81, whereinsaid starting material consists of a chain mixture in which at least 90%of said chains have the formula I, in which the uronic units are 40-60%consisting of iduronic acid.
 83. Process according to claim 81, wherein,in the formula I, n represents an integer from 3 to
 100. 84. Processaccording to claim 81, wherein said starting material consists of achain mixture in which at least 90% of said chains have the formula I′

in which the uronic units are 20-60% consisting of iduronic acid, q isan integer from 2 to 20 and the corresponding cation is chemically orpharmaceutically acceptable.
 85. Process according to claim 84, whereinsaid starting material consists of a chain mixture in which at least 90%of said chains have the formula I′, in which n is an integer from 3 to15.
 86. Process according to claim 81, wherein said starting materialconsists of a chain mixture in which the preponderant species has theformula I′a

in which the uronic units are 60-40% consisting of glucuronic acid and40% to 60% of iduronic acid, p is an integer from 4 to 8 and thecorresponding cation is chemically or pharmaceutically acceptable. 87.Process according to claim 86, wherein the mean molecular weight of saidstarting material is from approximately 2000 to approximately
 4000. 88.Process according to claim 86, wherein said starting material consistsof a chain mixture in which the preponderant species has the formula I′b

in which X is hydroxymethyl, m is 4, 5 or 6 and the glucuronic andiduronic units are present alternately, starting with a glucuronic oriduronic unit.
 89. Process according to claim 71, wherein said startingmaterial comes from a N-deacetylation and from a N-sulfation of a K5that is basically free of lipophilic substances.
 90. AnepiK5-N,O-oversulfate-derivative having an iduronic acid content of20-60%, a mean molecular weight from approximately 2,000 toapproximately 45,000 and a sulfation degree of at least 4, or one of itschemically or pharmaceutically acceptable salts, said derivative beingbasically inactive on the coagulation parameters.
 91. AnepiK5-N,O-oversulfate-derivative according to claim 90, whose meanmolecular weight is between approximately 15,000 and approximately45,000.
 92. An epiK5-N,O-oversulfate-derivative according to claim 90,whose mean molecular weight is between approximately 4,500 andapproximately 8,500.
 93. An epiK5-N,O-oversulfate-derivative accordingto claim 90, wherein said degree of sulfation is from 4 to 4.6.
 94. AnepiK5-N,O-oversulfate-derivative according to claim 90, which is 100%6-O-sulfated and 50-80% 3-O-sulfated in its glucosamine units, 5-10%O-monosulfated in glucuronic units, 10-15% 3-O-monosulfated in iduronicunits and 2,3-di-O-sulfated in the remaining uronic units.
 95. AnepiK5-N,O-oversulfate-derivative according to claim 90 consisting of achain mixture in which at least 90% of said chains have the formula III

in which the uronic units are 20-60% consisting of iduronic acid, R, R′,R″ represent hydrogen or SO₃ ⁻, R being SO₃ ⁻ in at least 40% of saidchain mixture, Z is a SO₃ ⁻ group, n is an integer from 2 to 100, thedegree of sulfation is at least 4 and the corresponding cation ischemically or pharmaceutically acceptable.
 96. AnepiK5-N,O-oversulfate-derivative according to claim 95, consisting of achain mixture in which at least 90% of said chains have the formula III,in which the uronic units are 40-60% iduronic acid.
 97. AnepiK5-N,O-oversulfate-derivative according to claim 95, consisting of achain mixture in which at least 90% of said chains have the formula III,in which n is an integer from 3 to
 100. 98. AnepiK5-N,O-oversulfate-derivative according to claim 95, which is aLMW-epiK5-N,O-oversulfate consisting of a chain mixture in which atleast 90% of said chains have the formula III′

in which the uronic units are 20-60% consisting of iduronic acid, q isan integer from 2 to 20, R, R′ and R″ represent hydrogen or a SO₃ ⁻group, Z is SO₃ ⁻, for a sulfation degree of from 4 to 4.6, and thecorresponding cation is one chemically or pharmaceutically acceptableion.
 99. A LMW-epiK5-N,O-oversulfate according to claim 98, consistingof a chain mixture in which at least 90% of said chains have the formulaIII′ in which q is an integer from 3 to
 15. 100. ALMW-epiK5-N,O-oversulfate according to claim 99, consisting of a chainmixture in which at least 90% of said chains have the formula III′ inwhich the uronic units are 40-60% consisting of iduronic acid.
 101. ALMW-epiK5-N,O-oversulfate according to claim 100, whose iduronic acidcontent is 50-55%.
 102. A LMW-epiK5-N,O-oversulfate according to claim98, consisting of a chain mixture in which at least 90% of said chainshave the formula III′ in which R is at least 40% SO₃ ⁻, R′ and R″ areboth SO₃ ⁻ or one is hydrogen and the other is 5-10% SO₃ ⁻ in glucuronicacid and 10-15% SO₃ ⁻ in iduronic acid.
 103. A LMW-epiK5-N,O-oversulfateaccording to claim 102, having a mean molecular weight fromapproximately 2,000 to approximately 16,000.
 104. ALMW-epiK5-N,O-oversulfate according to claim 103, having a molecularweight from approximately 4,500 to approximately 9,000.
 105. ALMW-epiK5-N,O-oversulfate according to claim 102, consisting of a chainmixture in which at least 90% of said chains have the formula III′ inwhich R is 50-80% SO₃ ⁻.
 106. A LMW-epiK5-N,O-oversulfate according toclaim 101, consisting of a chain mixture in which the preponderantspecies has the formula III′a

in which the uronic units are 20-60% consisting of iduronic acid, p isan integer from 4 to 8, Z is SO₃ ⁻, R, R′ and R″ are hydrogen or SO₃ ⁻,for a degree of sulfation from 4 to 4.6 and the corresponding cation ischemically or pharmaceutically acceptable.
 107. ALMW-epiK5-N,O-oversulfate according to claim 102, consisting of a chainmixture in which the preponderant species has the formula III′b

in which R, R′ and R″ are hydrogen or SO₃ ⁻, Z is SO₃ ⁻, X″ is OH orOSO₃ ⁻, m is 4, 5 or 6, for a degree of sulfation from 4 to 4.6, theglucuronic and iduronic units are present alternately, starting with aglucuronic or iduronic unit, and the corresponding cation is achemically or pharmaceutically acceptable ion.
 108. AnepiK5-N,O-oversulfate-derivative according to claim 90, wherein saidchemically or pharmaceutically acceptable salt is an alkaline metal,alkaline-earth metal, ammonium, (C₁-C₄)tetraalkylammonium, aluminum orzinc salt.
 109. An epiK5-N,O-oversulfate-derivative according to claim108, wherein said chemically or pharmaceutically acceptable salt is thesalt of sodium, calcium or tetrabutylammonium.
 110. AnepiK5-amine-O-oversulfate-derivative whose iduronic acid content is20-60% of the total of the uronic acids, having a mean molecular weightfrom approximately 3,500 to approximately 40,000 and a sulfation degreeof from 3.55 to 4, or one of its chemically or pharmaceuticallyacceptable salts.
 111. An epiK5-amine-O-oversulfate-derivative accordingto claim 110, consisting of a chain mixture in which at least 90% ofsaid chains have the formula II

in which the uronic units are 20-60% consisting of iduronic acid, n isan integer from 2 to 100, R, R′ and R″ are hydrogen or SO₃ ⁻, the degreeof sulfation is from 3.55 to 4 and the corresponding cation ischemically or pharmaceutically acceptable.
 112. AnepiK5-amine-O-oversulfate-derivative according to claim 111, of formulaII, wherein n represents an integer from 3 to
 100. 113. AnepiK5-amine-O-oversulfate-derivative according to claim 111, consistingof a chain mixture in which at least 90% of said chains have the formulaII in which the uronic units are 40-60% consisting of iduronic acid,with a mean molecular weight from approximately 2,000 to approximately40,000, R is at least 40%, SO₃ ⁻, R′ and R″ are both SO₃ ⁻ or one ishydrogen and the other is 5-10% SO₃ ⁻ in monosulfate glucuronic acid and10-15% SO₃ ⁻ in monosulfate iduronic acid.
 114. AnepiK5-amine-O-oversulfate-derivative according to claim 111, which is aLMW-epiK5-amine-O-oversulfate consisting of a chain mixture in which atleast 90% of said chains have the formula II in which the uronic unitsare 40-60% consisting of iduronic acid, R is at least 40%, SO₃ ⁻, R′ andR″ are both SO₃ ⁻ or one is hydrogen and the other is 5-10% SO₃ ⁻ inglucuronic acid and 10-15% SO₃ ⁻ in iduronic acid, n is an integer from3 to 15, with a mean molecular weight from approximately 4,000 toapproximately 8,000 and the corresponding cation is chemically orpharmaceutically acceptable.
 115. A LMW-epiK5-amine-O-oversulfateaccording to claim 134, consisting of a chain mixture in which thepreponderant species has the formula II′a

in which the uronic units are 20-60% consisting of iduronic acid, p isan integer from 4 to 8, R, R′ and R″ are hydrogen or SO₃ ⁻, bearing asulfated 2,5-anhydromannitol unit of structure (a′)

wherein R is hydrogen or SO₃ ⁻ at the reducing end of the majority ofsaid chains.
 116. A LMW epiK5-amine-O-oversulfate according to claim115, consisting of a chain mixture in which the preponderant species isa compound of formula II′b

in which the uronic units are 40-60% consisting of iduronic acid, m is4, 5 or 6, R, R′ and R″ are hydrogen or SO₃ ⁻, X″ is OH or OSO₃ ⁻, for asulfation degree of at least 3.4, the iduronic units being presentalternately, starting with a glucuronic or iduronic unit.
 117. ALMW-epiK5-N-sulfate virtually free of NH₂ and N-acetyl groups, having aniduronic acid content from 20 to 60% and a mean molecular weight fromapproximately 1,500 to approximately 12,000, or one of its chemically orpharmaceutically acceptable salts.
 118. A LMW-epiK5-N-sulfate accordingto claim 117, whose iduronic acid content is from 40 to 60% and the meanmolecular weight is from approximately 1,500 to approximately 10,000.119. A LMW-epiK5-N-sulfate according to claim 117, whose iduronic acidcontent is 50-55% and the mean molecular weight is from approximately1,500 to approximately 7,500.
 120. A LMW-epiK5-N-sulfate according toclaim 117, consisting of a chain mixture in which at least 90% of saidchains have the formula I′

in which the uronic units are 20-60% consisting of iduronic acid, q isan integer from 2 to 20, bearing a 2,5-anhydromanno unit of structure(a)

wherein X is formyl or hydroxymethyl, at the reducing end of themajority of said chains, and the corresponding cation is chemically orpharmaceutically acceptable.
 121. A LMW-epiK5-N-sulfate according toclaim 120, consisting of a chain mixture in which at least 90% of saidchains have the formula I′, in which the uronic units are 40-60%iduronic acid.
 122. A LMW-epiK5-N-sulfate according to claim 120,consisting of a chain mixture in which at least 90% of said chains havethe formula I′, in which n is an integer from 3 to
 15. 123. ALMW-epiK5-N-sulfate according to claim 120, consisting of a chainmixture in which the preponderant species has the formula I′a

in which the uronic units are 60-40% consisting of glucuronic acid and40% to 60% iduronic acid, p is an integer from 4 to 8 and thecorresponding cation is chemically or pharmaceutically acceptable. 124.A LMW-epiK5-N-sulfate according to claim 121, consisting of a chainmixture in which the preponderant species has the formula I′b

in which X is hydroxymethyl, m is 4, 5 or 6, the corresponding cation isa chemically or pharmaceutically acceptable ion and the glucuronic andiduronic units are present alternately, starting with a glucuronic oriduronic unit.
 125. A LMW-epiK5-N-sulfate according to claim 117,wherein said salt is selected from the group consisting of alkalinemetals, alkaline-earth metals, ammonium, (C₁-C₄)tetraalkylammonium,aluminum and zinc salts.
 126. A LMW-epiK5-N-sulfate according to claim125, wherein said salt is sodium, calcium or tetrabutylammonium salt.127. A process for the preparation of a LMW-epiK5-N-sulfate, whichcomprises subjecting a K5-N-sulfate, in any one order, (i) toC5-epimerization with a D-glucuronyl C5-epimerase isolated, purified andin solution or immobilized on a solid support, at a pH of approximately7, at a temperature of approximately 30° C. and for a time period of12-24 hours in the presence of at least one bivalent ion selected amongcalcium, magnesium, barium and manganese; and (ii) to nitrousdepolymerization optionally followed by reduction.
 128. Processaccording to claim 127, which is carried out in the order (i)-(ii). 129.Process according to claim 127, which is carried out in the order(ii)-(i).
 130. Process according to claim 129, wherein the productobtained upon termination of the depolymerization is a LMW-K5-N-sulfatewhich is directly subjected to C5-epimerization.
 131. Process accordingto claim 130, wherein said LMW-K5-N-sulfate has a mean molecular weightof more than 4,000.
 132. A pharmaceutical composition including, as anactive ingredient, a pharmacologically active amount of anepiK5-N,O-oversulfate-derivative according to claim 90, in mixture witha pharmaceutical excipient.
 133. A cosmetic composition including aneffective amount of an epiK5-N,O-oversulfate-derivative according toclaim 90, in mixture with a cosmetic excipient.
 134. ALMW-epiK5-amine-O-oversulfate consisting of mixture of chains in whichat least 90% of said chains have the formula II′

in which 20-60% of the uronic acid units are those of iduronic acid, qis an integer from 2 to 20, R, R′ and R″ are hydrogen or SO₃ ⁻, bearinga sulfated 2,5-anhydromannitol unit of structure (a′)

wherein R is hydrogen or SO₃ ⁻, at the reducing end of the majority ofsaid chains, for a sulfation degree of at least 3.4, and thecorresponding cation is a chemically or pharmaceutically acceptable ion.